Summary: This area of my research tests the hypothesis that environmental exposures produce specific patterns of gene mutation in human tumors. Such patterns can be used both to identify critical target genes and to suggest mutational mechanisms by which an environmental agent causes cancer. If specific carcinogens produce characteristic patterns of gene mutation in tumors, the detection of those patterns would be a powerful tool in studies of environmental risk and for use in prevention and early diagnosis. In recent years we have begun to extend this concept in our ongoing molecular epidemiologic and clinical studies designed to look at germline mutation and, using special techniques that we have developed, to look at DNA damage in very small samples of preneoplastic and normal tissue. With the establishment of the Comet assay in the lab, a technique that allows us to measure general DNA damage in individual living cells, we are now developing a new clinical-experimental study where we plan to measure levels of DNA damage in sequential biopsies of colon epithelium from people as we subject them to different dietary regimes. A long term goal is to develop a quantitative measure of the level of DNA mutation in normal tissue or "somatic mutational load". Such a metric could provide a tissue specific measure of lifetime environmental exposure, integrated across diet, genetic susceptibility, and repair, and might offer a more precise estimate of risk for cancer, neurologic, reproductive, and other diseases where DNA damage plays a role. Fluorescence Bronchoscopy and Molecular Characterization of Abnormal Bronchial Lesions (LIFE Study): Our major study that is currently in the clinic is designed to test the hypothesis that exposure correlates with the pattern of mutation in premalignant and normal lung tissues and that such mutations may have prognostic significance for lung cancer development. We are using the Lung Imaging Fluorescent Endoscope (LIFE), a newly developed bronchoscopy technique to collect normal, premalignant, and neoplastic tissue samples from patients at high risk of lung cancer from smoking, occupational exposures, or because of family history. These people are followed over a 2 year period with repeat bronchoscopies and biopsy allowing us to follow the molecular changes in individual lesions over time. In addition we have a small pilot project, jointly funded with UNC, to obtain optimally-collected tumor and normal tissue from patients undergoing thoracotomy for lung cancer. Last Year's Progress: LIFE lung cancer study: We have enrolled and bronchoscoped an additional 12 patients in the last year bringing our current total to 40 patients, many of whom have undergone second bronchoscopies. We use laser capture microdissection (LCM) of frozen biopsies to collect specific cells, and have developed special PCR methods that allow us to work with only 50 cells in order to examine loss of heterozygosity (LOH) at a panel of selected loci. In addition, cells grown in culture from these patients are being examined for cytogenetic abnormalities, telomerase activity, and will be examined immunohistochemically for p53 and p21. Preliminary results on LOH, cytogenetic abnormalities, and telomerase activity are being provided from this work in three separate presentations at the 10th World Conference on Lung Cancer this summer. Environmental exposure and p53 mutation patterns in bladder cancer: We have recently had our manuscript of our molecular epidemiology study of the causes and mechanisms of bladder cancer accepted for publication in Cancer Research.. We utilized our previously-generated data on carcinogen metabolism gene polymorphisms along with new p53 mutation analyses of 143 paraffin-embedded tumors. Using a case-control design we sought evidence of environmental, occupational, and tobacco mutagens in the mutation spectrum and mechanistic clues from associations of mutation subsets with metabolic genotypes. The principal observation is that GC>AT transitions at CpG dinucleotides occur significantly more often in tumors from people with environmental exposure, particularly smokers, than those without exposure. Coupled with evidence of a coding strand bias, and a possible association with NAT2 slow acetylator genotype, this is perhaps the first plausible evidence of a tobacco signature mutation in bladder cancer. Germline mutations in Chernobyl cleanup workers: In this recently completed molecular-epidemiology study we tested the hypothesis that Chernobyl accident cleanup workers had higher rates of germline mutations after their exposure than before their exposure. We compared rates of DNA microsatellite and minisatellite mutation in children conceived before their exposure to children conceived after their exposure. We demonstrate a relatively high rate of germline mutation in mini and microsatellite loci, but show no significant difference in rates between children conceived prior to their father's exposure vs those conceived post exposure. This manuscript is currently being revised for resubmission to Mutation Research. Cadmium mismatch repair inhibition, and microsatellite mutation: We entered into a collaboration with Mike Resnick and Tom Kunkel to investigate the effect of cadmium on mismatch repair. We extended out technique for amplifying small quantities of DNA down to the single molecule level in order to evaluate whether human cells grown in culture with environmentally-relevant concentrations of cadmium had increase mutation rates in microsatellite sequences. Preliminary results of this work were included in the resulting Nature Genetics paper. We are hoping to extend this work when a new postdoctoral fellow is hired to replace Dr. Slebos (now at Vanderbilt U).